Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of in-vitro incorporation of [3H]thymidine into hamster follicles during the oestrous cycle

Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per microgram follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7-8 layers granulosa cells plus theca). When expressed as thymidine per follicle or microgram DNA, there was a significant increase in incorporation for stages 1-4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per microgram DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1-4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.     

In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin

Hamster ovarian follicles at Stages 1 to 10 (Stages 1-4: follicles with 1-4 layers of granulosa cells (GC); Stages 5-7: 5-10 layers GC plus theca; Stages 8-10: antral follicles) were isolated on the morning of proestrus or estrus and incubated for 2 h in the absence or presence of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl), progesterone (P4), 17 alpha-hydroxyprogesterone (17OHP), or androstenedione (A). Steroid accumulations in the media were measured by radioimmunoassay (RIA). On proestrus, without any hormonal stimulus, consistent accumulation of P4 through estradiol-17 beta (E2) occurred in low amounts only from Stage 6 and on; both FSH (5-25 ng) and LH (1-25 ng) significantly stimulated steroidogenesis by Stage 6-10 follicles, and the effects of FSH, except for Stage 10, were largely attributable to LH contamination. However, 25 ng FSH significantly stimulated A production by Stages 1-4, whereas 1-25 ng LH was ineffective. On estrus, follicles at all stages, especially 1-6, showed significant and dose-dependent increases in P4 production in response to FSH; both FSH and LH significantly stimulated P4 and 17OHP accumulation from Stage 5 onwards; however, there was no increase in A and E2 compared to controls. Even the smallest estrous follicles showed a shift to predominance of P4 accumulation. On proestrus, Prl had a negative influence on LH-induced accumulation of P4 and 17OHP by Stages 7-9 and 6-8, respectively, without affecting A or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of unilateral ovariectomy on follicular development in the cyclic hamster

Hamsters were hemispayed at 09:00 h on Day 3 of the cycle (Day 1 equal to ovulation) and were killed 1 h after injection of [3H]thymidine at 09:00, 12:00, 17:00 or 22:00 h. Unilateral ovariectomy had no effect on the number of Stage 1 or Stage 2 follicles, but there were significantly fewer Stage 3 follicles between 10:00 and 13:00 h. This decrease was not encountered in intact hamsters and was reflected in an increase in the number of Stage 6 (antral) follicles. At 13:00 h there was no difference in the number of atretic follicles between the experimental and control groups. It is concluded that preantral follicles with 6-7 layers of granulosa cells were recruited within 4 h after unilateral ovariectomy and transformed into antral follicles.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Changes in inhibin activity, and progesterone, oestrogen and androstenedione concentrations, in rat follicular fluid throughout the oestrous cycle

Follicular fluid was aspirated from all visible surface follicles of rats at selected times of the oestrous cycle. Fluids from a pair of rat ovaries were pooled and assayed for inhibin activity by the rat anterior pituitary cell culture assay. Serum LH, FSH and progesterone as well as follicular fluid progesterone, total oestrogens and androstenedione were also measured. Follicular fluid inhibin activity was relatively constant throughout the oestrous cycle (30.7 +/- 3.4% inhibition of FSH per 0.1 microliter follicular fluid) except for a well defined surge at pro-oestrus (09:00-16:00 h, peak at 14:00 h = 84.0 +/- 7.2% inhibition of FSH per 0.1 microliter follicular fluid). The follicular fluid was not treated with charcoal before assay because a pilot experiment showed that such treatment did not alter the inhibin activity of follicular fluid. Steroids in follicular fluid were generally lowest on the afternoon of oestrus and the morning of dioestrus I and generally elevated during pro-oestrus.     

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.     

Genetic differences in follicular DNA synthesis between two strains of hamsters

This study was designed to compare our previous results on ovarian follicular DNA synthesis by hamsters obtained from Sasco Laboratories with a different breeding colony: Harlan. Follicles from proestrous Harlan hamsters required twice as much [3H] thymidine and a minimum of 4 hr of in vitro exposure to 100 ng of ovine follicle-stimulating hormone (FSH) before a significant increase in DNA synthesis was elicited compared with 30-120 min for the Sasco breed. Peak responsiveness to FSH was observed at 8-hr incubation for the Harlan strain with significant increases in DNA per follicle at 8-12 hr. Both strains increased DNA synthesis with as little as 25 ng of ovine FSH and the response was elicited in all growing follicles, from preantral stages with one to four layers of granulosa cells, lacking theca (Stages 1-4) to mature antral follicles (Stages 8-10). A recombinant bovine FSH, devoid of luteinizing hormone activity, was not as effective as ovine FSH (which has 4% luteinizing hormone contamination) in stimulating DNA synthesis by large preantral and antral follicles. In vitro responsiveness to ovine FSH was abolished in the absence of Ca2+ in the culture medium and 0.05 mM Ca2+ was the optimal amount. For both strains of hamsters, the highest rate of DNA synthesis in response to endogenous gonadotropins was on the morning of estrus--when the second surge of FSH was in progress--and Harlan follicles in vitro also showed maximal stimulation by FSH on this day. Where the two strains differed was that the Harlan strain did not show an increase in follicular DNA synthesis on the afternoon of proestrus--when the preovulatory increase in gonadotropins commenced. When expressed as DNA per follicle, DNA approximately doubled from Stages 1 to 5 and then entered a new growth phase at Stage 6 (large preantral follicles) with a steeper increase. Collectively, these experiments show that strain characteristics can alter the latency and degree of follicular DNA replication in response to endogenous or exogenous FSH.     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.     

Influence of short days on diurnal patterns of serum gonadotrophins and prolactin concentrations in the male Djungarian hamster, Phodopus sungorus

Exposure to short days for 8 weeks suppressed mean serum concentrations of FSH, LH and prolactin compared to hamsters kept in long days. Hamsters in short days exhibited a small afternoon rise in serum FSH, but serum LH and prolactin did not exhibit 24-h variations. In hamsters under long days, a late afternoon-early evening increase was evident for circulating prolactin but none was detected for the gonadotrophins. A fall in testes weights rapidly occurred by 14-28 days after transfer to short days. This was accompanied or preceded by a decrease in serum gonadotrophins and prolactin. Reductions in serum FSH and LH occurred in short days in blood samples taken at 09:00 h or 15:00 h. However, the nadir in serum prolactin was first achieved (at 09:00 h), at least 7 days before that at 15:00 h (i.e. Day 14 versus Day 21 of short photoperiod, respectively). The ability to secrete gonadotrophins was further tested in hamsters that had undergone gonadal regression. Castration of hamsters exposed to short days or injected with melatonin in the afternoon, a treatment known to mimic short day effects, induced a 3- to 5-fold increase in serum gonadotrophins. However, this rise in FSH and LH was significantly attenuated compared to the 10-fold response in controls in long days. The results indicate that gonadal involution induced by short days may be mediated by the decline in mean gonadotrophin secretion which, in turn, is regulated by responsiveness to steroids, as well as a mechanism independent of the negative feedback action of gonadal steroids.     

Quantitative autoradiographic study of FSH binding sites in prepubertal ovaries of three strains of rats

Iodinated FSH was injected to 18- and 36-day-old rats of 3 strains (03, 04 and 12) with different sensitivity to FSH (12 less than 03 less than 04) and autoradiography was performed on histological sections of the labelled ovaries. Specific labelling was quantified by microphotometry on histological slides, on granulosa cells of individual follicles with different sizes (greater than 80 micron diameter) and qualities. In small preantral follicles (less than 160 micron diameter) the labelling was low and homogeneous within the granulosa; it increased between 18 and 36 days of age in the 3 strains. At 36 days, ovaries were characterized by the presence of large preantral and antral follicles with a higher labelling in the outer layers of granulosa (near the theca), compared to the inner layers. In definitely atretic follicles, a loss of binding sites was detected in the outer layers. In rats of Strains 03 and 04, the number of binding sites for FSH in the outer layers of granulosa of follicles with a diameter of greater than 160 micron increased with follicular size; no change was detected in follicles of Strain 12 rats. The low number of binding sites for FSH and the lack of terminal maturation which characterize the follicles of strain 12 rats can be related to the poor and delayed follicular development, the low sensitivity to exogenous FSH and the low fertility of the animals of this strain.     

Prolactin and somatotropin binding by granulosa cells of cows at different reproductive states

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3-5 to 6-10 mm, specific binding of 125I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data suggest that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1-2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Changes in rat ovaries of specific binding for LH, FSH and prolactin during the oestrous cycle and pregnancy.

In cyclic rats, the highest ovarian specific binding for LH was 6-0+/- 2-2% inpro-oestrus. During pregnancy, the specific binding of 125I-labelled bovine LH by rat ovaries increased gradually and reached a maximum of 24-1+/-4-9% between Days 14 and 18 of gestation; a slight decrease in binding was observed at Day 20 of pregnancy. Ovarian specific binding for FSH was also highest in pro-oestrus (8-9+/-2-1%), decreasing to about 50% in oestrus and metoestrus, but staying relatively constant during pregnancy. For prolactin, the specific binding in rat ovaries was highest (7-1+/-1-6%) in pro-oestrus, quite high in metoestrus and dioestrus and low in oestrus. Specific binding increased gradually only after Day 14 of pregnancy. Serum concentrations of rat LH, FSH and prolactin at different stages of the oestrous cycle and during pregnancy were determined by radioimmunoassays, and no obvious correlation was observed between levels of circulating hormones and the specific binding of these hormones in ovarian tissues. Affinity constants (Ka) for the hormones were very similar between ovaries from pro-oestrous rats and late-pregnant rats, being 0-31 X 10(9) M-1 for LH, 0-65 X 10(10)M-1 for FSH, and 1-14 X 10(10)M-1 for prolactin. Increases in specific binding for different hormones were due to increases of total binding sites in the ovary under different physiological states.     

Effect of pentobarbitone sodium and bromocriptine on follicular oestradiol production in the rat

Injection of an ovulation-blocking dose of pentobarbitone sodium given in the early afternoon of pro-oestrus in rats decreased follicular oestradiol production in vitro the next day (2.42 +/- 0.11 ng/4 h/follicle in pro-oestrous rats, 0.49 +/- 0.04 ng/4 h/follicle in pentobarbitone-treated rats). Pentobarbitone, given 1 day earlier (at dioestrus II), prevented the increase in oestradiol production that normally occurs between di-oestrus II and pro-oestrus. Injection of a subovulatory amount of hCG (0.5 i.u.) given after pentobarbitone injection inhibited the decrease in follicular oestradiol production induced by pentobarbitone. The pentobarbitone-induced decrease in oestradiol production was also prevented by bromocriptine (1 mg) given at di-oestrus II (15:00 h) and pro-oestrus (09:00 h). Bromocriptine is an effective inhibitor of prolactin secretion and this suggests therefore that the decrease in follicular oestradiol production after pentobarbitone is due to the preovulatory surge of prolactin. However, pretreatment with bromocriptine also inhibited the effect of pentobarbitone on oestradiol production when pentobarbitone was given at di-oestrus II. Moreover, when ergocornine (another inhibitor of prolactin secretion) was used instead of pentobarbitone to block ovulation, follicular oestradiol production was also decreased the next day. In contrast to bromocriptine, ergocornine was not able to prevent the pentobarbitone-induced decrease in follicular oestradiol production. These results indicate that the decrease in follicular oestradiol production after pentobarbitone injection is due to inhibition of the serum concentrations of LH rather than the preovulatory surge of prolactin. How bromocriptine (but not ergocornine) prevents the pentobarbitone-induced decrease in oestradiol production is not clear.     

Exogenous progesterone reduces follicular prostaglandin E and 6-keto prostaglandin F-1 alpha in the cyclic hamster

In cyclic hamsters, exogenous progesterone (100 micrograms) administered s.c. at 09:00 h on the day of dioestrus II reduced prostaglandin (PG) E and 6-keto PGF-1 alpha but not PGF concentrations in preovulatory follicles measured at 09:00 h of pro-oestrus. The injection of 10 micrograms ovine LH (NIADDK-oLH-25) concurrently with 100 micrograms progesterone on dioestrus II prevented the decline in follicular PGE and 6-keto PGF-1 alpha values. Administration of LH alone did not significantly alter follicular PG concentrations. Inhibition of follicular PGE accumulation by progesterone was due to a decline in granulosa PGE concentration and not thecal PGE. Progesterone administration also reduced follicular oestradiol concentrations. Administration of oestradiol-17-cyclopentanepropionate (ECP) (10 micrograms) with progesterone did not prevent the decline in follicular PGE and 6-keto PGF-1 alpha but did increase follicular PGF concentrations. However, ECP given alone on dioestrus II reduced follicular PGE and increased PGF concentrations in preovulatory follicles on pro-oestrus. It is concluded that exogenous progesterone administered on dioestrus II inhibits granulosa PGE and 6-keto PGF-1 alpha accumulation in preovulatory follicles, probably by reducing serum LH concentrations, and that the granulosa cells, which are LH-dependent, are a major source of follicular PGE.     

Changes in the density of peripheral benzodiazepine binding sites in genital organs of the female rat during the oestrous cycle

The affinity and the density of peripheral-type benzodiazepine binding sites (PBzS) in tissues from the genital organs of female rats were studied during the oestrous cycle. When comparing PBzS density on the day of oestrus to PBzS density on the day of pro-oestrus, a significant increase was observed in the ovary (1.9-fold), oviduct (2.4-fold) and uterus (1.7-fold), but not in the kidney. Serum oestradiol also increased to a maximum on the day of pro-oestrus. The ovarian and uterine PBzS density and serum concentrations of oestradiol and progesterone were measured every 8 h between the days of dioestrus and pro-oestrus. Ovarian and uterine PBzS density increased to a maximal value at 01:00 and 09:00 h, respectively, on the day of pro-oestrus. However, a significant increase in PBzS density occurred in the ovary (P less than 0.02) and uterus (P less than 0.001) at 09:00 h on the day of pro-oestrus as compared to 09:00 h on the day of dioestrus. These changes were associated with an increase in serum oestradiol and progesterone concentrations. The affinity of PBzS in all tissues examined remained unaltered during the oestrous cycle. This study demonstrates that changes associated with the oestrous cycle occur in the density of PBzS in various genital organs.     

Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P < 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles > 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Stimulatory and inhibitory effects of progesterone on follicular development in the hypophysectomized follicle-stimulating hormone/luteinizing hormone-treated hamster

Cyclic hamsters hypophysectomized at estrus (Day 1 of the cycle) and injected with 5 micrograms follicle-stimulating hormone (FSH) on Day 1 and 20 micrograms luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) from Days 1-4 ovulated 15.3 ova, in response to 30 IU human chorionic gonadotropin (hCG) administered at 1500 h on Day 4 (Kim and Greenwald, 1984). When 1 mg progesterone (P4) was administered daily from Days 1-4 concurrent with the above regimen, ovulation increased to 38 ova, a clearcut superovulatory response. However, daily injection of 1, 10, or 100 micrograms P4 plus FSH and LH reduced the number of antral follicles present on the afternoon of Day 4 to 3-4 per ovary, compared to 9 per ovary after FSH-LH alone, and the ovulation rate was drastically reduced with most animals being anovulatory. Substituting 1 mg 17 alpha-hydroxyprogesterone or estradiol cyclopentylpropionate for P4 on Days 1-4 did not alter the number of antral follicles on Day 4 from FSH-LH alone, whereas 1 mg androstenedione or 1 mg testosterone cyclopentylpropionate reduced the number of antral follicles to 3 or less. Hence, the stimulatory effects of 1 mg P4 are not attributable to its conversion to other P4 derivatives. After the concurrent injection of 1 mg P4 and FSH-LH, on the afternoon of Day 3, an average of only 1.8 large preantral follicles was present per ovary. By the morning of Day 4, however, the ovary contained 14 large preantral and early antral follicles in addition to 8 large antral follicles. Injection of hCG at this time resulted in the ovulation of 14.5 ova.(ABSTRACT TRUNCATED AT 250 WORDS)